To reduce high attrition rates and accelerate discovery and development timelines, the pharmaceutical industry seeks in vitro alternatives to interrogate drug candidates prior to animal, humanized animal, and human studies. Enormous cost, ethical concerns and increased pressure from regulatory agencies all drive demand for cell-based models and perfusion methodologies that can be used for in vitro screening of both large and small molecule drugs, and to triage toxic and ineffective leads earlier, prior to in vivo studies. However, it has been difficult finding a cell-based model that mimics function of living tissues, and a corresponding perfusion system which mimics both the function of vasculature and in vivo tissue perfusion for physiologically closer tissue modeling, tissue metabolic sustenance, and the delivery, distribution and testing of compounds within the interior of tissue mimicking cell mass.
Recently, three-dimensional (3D) cell cultures have emerged as an alternative to screening in a flat layer of cells as means to model tissues with improved physiological relevance for biomedical research and in vitro drug testing of all stages. 3D cultures are cellular networks organized in three dimensions—an environment that is much more similar to that found in vivo. Examples include 3D cell cultures grown in extracellular matrix (ECM) gels or gels mimicking the ECM; 3D cell aggregates such as tumor spheroids, embryoid bodies and hanging drop cell cultures; and cultures grown in 3D rigid scaffolds, among others.
For most drug screening applications, mature, tissue-like 3D cell cultures with well developed cell networks, cell-cell and cell-ECM signaling and interactions are needed. However, it has been challenging to grow and maintain such cultures due to diffusive mass transport limitations within their interior, and especially in the case of a common 3D ECM gel culture model or a high-cell-density spheroid model. Accordingly, without functional vasculature and non-invasive yet efficient intra-3D-culture perfusion, it has been difficult to deliver and distribute nutrients, drugs and other compounds intra-3D-culture for physiologically closer tissue modeling and drug testing in the same. The latter is especially true for large molecule drugs (e.g. monoclonal antibodies, therapeutic proteins, tissue growth factors, etc.) and drugs which mass transport in vivo relies on convection.
In standard, diffusion-limited culturing systems in vitro, 3D cultures which model tissues perfused in vivo suffer from the same problems as do slices of said explanted tissue. Tissues and tissue-like 3D gel cultures pose high resistance to mass transport due to steric hindrance and ionic interactions between solutes and ECM gel constituents. This reduces molecular diffusivities by approximately 18% to 93% from free solution values [Swartz, M. A., and Fleury, M. E. 2007. Annu Rev. Biomed. Eng. 9: 229-256]. From the 3D culture periphery to its interior, intra-culture availability of nutrients and gas reduces and metabolic waste accumulates, leading to formation of a necrotic, inner core. Accordingly, in mature 3D cultures, the necrosis and inconsistent decay that progresses culture-to-culture calls into question the reproducibility of cell outcomes and interpretation of screening results (cultures decay before studies are completed, adhere poorly, begin to float, or are simply aspirated during media exchanges in standard protocols).
Prior art 3D cell culture perfusion tools have also been riddled with problems. Lack of intra-culture perfusion methods (intra-gel or through a dense mass of cells); cumbersome priming and out-gassing; pressure surges and clogging of miniaturized components, complex setup, frequent user interventions in operation with overall poor culture performance relative to user input and cost have been some of the problems even when throughput was low. In part, this is because these tools were originally developed for perfusion of cell monolayer cultures, and then applied with little modification to perfusion of 3D cultures. For example, in a two-dimensional (2D) cell culture having one layer of cells, gel is typically absent. However, perfusion intra-gel is necessary if cells are to be perfused in a 3D gel cell culture plug.
Since prior art flow geometry has rarely been optimized for intra-3D-culture perfusion forcing flow around 3D culture rarely made flow of concentrated solutions pass intra-3D-culture (due to higher culture resistance), and the dominant mode of intra-3D-culture mass transport has remained diffusion-limited. The common prior art “superfusion” approach succeeded mainly to efficiently mix, stir or otherwise maintain high nutrient and gas concentration in the medium that surrounds the culture. Accordingly, forcing flow intra-culture (e.g. 3D cell culture gel plug) and forcing it non-invasively has remained a delicate task.
Another frequently overlooked problem in prior art cell culture perfusion, whether with 2D cultures or 3D cultures, is that the vast majority of perfusion tools have been based on unidirectional perfusion. In this perfusion setting, signaling molecules cells secrete to regulate their environment, growth and many other functions have been constantly washed away with the one-way flow. This is of concern because trophic factors, autocrine and paracrine signaling molecules cell secrete, need, and are surrounded by, are critical for sustained 3D cell culture growth, function, and drug testing. Although medium recycling has been used to mitigate this effect, relatively high liquid volume to culture volume in a single loop have generally contributed to dilution and delay in bringing these molecules back to cells.
Each of these issues likely contributes to the findings of the Comley article [Comley, D. J., 2010. Drug Discovery World. 11(3): 25-41] relating to the lack of adequate perfusion methods and tools to support 3D cell cultures in high-throughput screening and high-content screening applications. Specifically, in discussing state of the art in 3D cell culture perfusion tools the Comley article stated “Currently they are optimised to quite specific 3D applications (e.g. invasion assay profiling) or to support specific tumour cell models.”
Accordingly, what is needed in the art are methods and tools dedicated to 3D cell culture perfusion, to force flow intra-cultures non-invasively and without abrupt fluctuations; with delivery and distribution of nutrients, gas, and test compounds within the 3D cultures in a manner analogous to the in vivo situation at a minimal loss of cell secreted molecules; and in high-throughput. More specifically, what is needed are (A) materials which mimic function of in vivo vasculature to efficiently deliver and distribute agents to the interior of tissue mimetic cultures in high-throughput; materials which mimic the function of vasculature in unperfused cultures under hydrostatic and osmotic pressure differences; materials which mimic the function of vasculature in forced convection intra-culture perfusion; (B) methods of making synthetic vasculature and methods of controlling the synthetic vasculature volume fraction in a 3D culture volume, capillary diameters and inter-capillary distances; methods of using said vasculature to vascularize cell cultures and to deliver and distribute soluble factors and gas; (C) perfusion methods and tools to non-invasively deliver and distribute agents intra-culture without abrupt fluctuations in culturing conditions, and without prohibitively high shear and normal stresses causing cellular injuries; perfusion tools and methods which prevent loss of cell signaling molecules while removing waste products; perfusion tools and methods that enable easy plating of cultures, minimize paths of low resistance around the cultures, and evacuate air in high-throughput manner; and (D) methods of making and using perfusion platforms and their integrative platform variations (which meet A-C) in a high-throughput format for ease of automation and user adoption.